Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP enhances the growth and osteolytic potential of prostate cancer cells, it is important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) downregulates PTHrP expression in a variety of human cell types, including prostate cancer cells. Therefore, downregulation of PTHrP gene expression by 1,25(OH)2D3 may enhance the therapeutic benefits of 1,25(OH)2D3 by neutralizing PTHrP’s contribution to the pathogenesis and progression of prostate carcinoma and its tendency to metastasize to bone. In this study, we show that 1,25(OH)2D3 and its non-hypercalcemic analog EB1089 decrease cell proliferation and suppress PTHrP mRNA and protein levels in the human prostate cancer cell lines LNCaP, C4-2, and PC-3. These cell lines represent early to advanced stages of prostate cancer, since LNCaP cells are weakly metastatic, androgen receptor (AR)-positive and androgen-responsive cells, C4-2 cells are AR-positive and androgen-independent LNCaP variants, and PC-3 cells are highly metastatic AR-negative cells. We identified a negative vitamin D response element (nVDREhPTHrP) within the human PTHrP gene; interaction of the vitamin D receptor (VDR) with this nVDRE is decreased by 1,25(OH)2D3. However, transient transfection of nVDREhPTHrP cloned upstream of the SV40 promoter downregulated promoter activity in response to 1,25(OH)2D3 or EB1089 treatment in LNCaP and C4-2, but not in PC-3, cells. The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXR&#945; forms part of the nuclear protein complex that interacts with nVDREhPTHrP with the VDR in LNCaP, C4-2, and PC-3 cells. The RXR ligand 9-cis-retinoic acid (9-cis-RA) downregulates PTHrP mRNA levels in both LNCaP and C4-2 cells; this decrease is more pronounced in LNCaP than in C4-2 cells. 9-cis-RA also enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect is more pronounced in LNCaP cells. Co-treatment with 1,25(OH)2D3 or EB1089 and 9-cis-RA further decreased promoter activity driven via nVDREhPTHrP in LNCaP, but not C4-2, cells. The proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-RA. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell line-specific manner in prostate cancer cells.